ABSTRACT
In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.
Subject(s)
Chromosomes, Mammalian , Genetic Therapy/methods , Granulocytes/metabolism , Granulomatous Disease, Chronic/therapy , Hypoxanthine Phosphoribosyltransferase/genetics , Induced Pluripotent Stem Cells/metabolism , NADPH Oxidase 2/genetics , Aminopterin/metabolism , Aminopterin/pharmacology , Animals , Base Sequence , CRISPR-Cas Systems , Cell Differentiation , Clone Cells , Culture Media/chemistry , Disease Models, Animal , Gene Editing/methods , Granulocytes/cytology , Granulocytes/drug effects , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/metabolism , Granulomatous Disease, Chronic/pathology , Humans , Hypoxanthine/metabolism , Hypoxanthine/pharmacology , Hypoxanthine Phosphoribosyltransferase/deficiency , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/pathology , Male , Mice , NADPH Oxidase 2/deficiency , Proof of Concept Study , Sequence Deletion , Thioguanine/metabolism , Thioguanine/pharmacology , Thymidine/metabolism , Thymidine/pharmacology , X Chromosome/chemistry , X Chromosome/metabolismABSTRACT
Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27â¯months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Motor Neurons/pathology , Muscular Diseases/genetics , Nerve Degeneration/genetics , Superoxide Dismutase-1/genetics , TDP-43 Proteinopathies/genetics , Amyotrophic Lateral Sclerosis/genetics , Animals , Animals, Genetically Modified , Disease Models, Animal , Humans , Muscular Diseases/pathology , Nerve Degeneration/pathology , Swine , TDP-43 Proteinopathies/pathologyABSTRACT
Hepatocellular carcinoma (HCC) is a frequent neoplasia and a leading cause of inflammation-related cancer mortality. Despite that most HCCs arise from persistent inflammatory conditions, pathways linking chronic inflammation to cancer development are still incompletely elucidated. We dissected the role of adaptive immunity in the Mdr2 knockout (Mdr2-/- ) mouse, a model of inflammation-associated cancer, in which ablation of adaptive immunity has been induced genetically (Rag2-/- Mdr2-/- and µMt-Mdr2-/- mice) or with in vivo treatments using lymphocyte-specific depleting antibodies (anti-CD20 or anti-CD4/CD8). We found that activated B and T lymphocytes, secreting fibrogenic tumor necrosis factor alpha (TNFα) and other proinflammatory cytokines, infiltrated liver of the Mdr2-/- mice during chronic fibrosing cholangitis. Lymphocyte ablation, in the Rag2-/- Mdr2-/- and µMt-Mdr2-/- mice, strongly suppressed hepatic stellate cell (HSC) activation and extracellular matrix deposition, enhancing HSC transition to cellular senescence. Moreover, lack of lymphocytes changed the intrahepatic metabolic/oxidative state, resulting in skewed macrophage polarization toward an anti-inflammatory M2 phenotype. Remarkably, hepatocarcinogenesis was significantly suppressed in the Rag2-/- Mdr2-/- mice, correlating with reduced TNFα/NF-κB (nuclear factor kappa B) pathway activation. Ablation of CD20+ B cells, but not of CD4+ /CD8+ T cells, in Mdr2-/- mice, promoted senescence-mediated fibrosis resolution and inhibited the protumorigenic TNFα/NF-κB pathway. Interestingly, presence of infiltrating B cells correlated with increased tumor aggressiveness and reduced disease-free survival in human HCC. CONCLUSION: Adaptive immunity sustains liver fibrosis (LF) and favors HCC growth in chronic injury, by modulating innate components of inflammation and limiting the extent of HSC senescence. Therapies designed for B-cell targeting may be an effective strategy in LF. (Hepatology 2018;67:1970-1985).
Subject(s)
B-Lymphocytes/immunology , Carcinogenesis/immunology , Carcinoma, Hepatocellular/immunology , Liver Cirrhosis/immunology , Liver Neoplasms/immunology , ATP Binding Cassette Transporter, Subfamily B/genetics , Adaptive Immunity/immunology , Animals , Carcinogenesis/pathology , Cell Culture Techniques , Cellular Senescence/immunology , Cytokines/metabolism , Disease Models, Animal , Humans , Immunohistochemistry , Liver/immunology , Liver/pathology , Liver Cirrhosis/pathology , Liver Neoplasms/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
The immune and the skeletal system are tightly interconnected, and B lymphocytes are uniquely endowed with osteo-interactive properties. In this context, receptor activator of NF-κB (RANK) ligand (RANKL) plays a pivotal role in lymphoid tissue formation and bone homeostasis. Although murine models lacking RANK or RANKL show defects in B cell number, the role of the RANKL-RANK axis on B physiology is still a matter of debate. In this study, we have characterized in detail B cell compartment in Rankl(-/-) mice, finding a relative expansion of marginal zone B cells, B1 cells, and plasma cells associated with increased Ig serum levels, spontaneous germinal center formation, and hyperresponse to CD40 triggering. Such abnormalities were associated with an increased frequency of regulatory B cells and augmented B cell-derived IL-10 production. Remarkably, in vivo IL-10-R blockade reduced T cell-triggered plasma cell differentiation and restrained the expansion of regulatory B cells. These data point to a novel role of the RANKL-RANK axis in the regulation of B cell homeostasis and highlight an unexpected link between IL-10 CD40 signaling and the RANKL pathway.
Subject(s)
B-Lymphocytes/immunology , Interleukin-10/immunology , RANK Ligand/deficiency , RANK Ligand/immunology , Animals , Mice , Mice, KnockoutABSTRACT
Autosomal recessive osteopetrosis is usually associated with normal or elevated numbers of nonfunctional osteoclasts. Here we report mutations in the gene encoding RANKL (receptor activator of nuclear factor-KB ligand) in six individuals with autosomal recessive osteopetrosis whose bone biopsy specimens lacked osteoclasts. These individuals did not show any obvious defects in immunological parameters and could not be cured by hematopoietic stem cell transplantation; however, exogenous RANKL induced formation of functional osteoclasts from their monocytes, suggesting that they could, theoretically, benefit from exogenous RANKL administration.
Subject(s)
Osteopetrosis/genetics , RANK Ligand/genetics , Animals , Consanguinity , Female , Genes, Recessive , Humans , Male , Mice , Osteoclasts , PedigreeABSTRACT
Cornelia de Lange syndrome is a multisystem developmental disorder characterized by facial dysmorphisms, upper limb abnormalities, growth delay and cognitive retardation. Mutations in the NIPBL gene, a component of the cohesin complex, account for approximately half of the affected individuals. We report here that mutations in SMC1L1 (also known as SMC1), which encodes a different subunit of the cohesin complex, are responsible for CdLS in three male members of an affected family and in one sporadic case.
Subject(s)
Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , De Lange Syndrome/genetics , Genetic Diseases, X-Linked/genetics , Mutation , Female , Humans , Male , PedigreeABSTRACT
Omenn syndrome (OS) is an atypical primary immunodeficiency characterized by severe autoimmunity because of activated T cells infiltrating target organs. The impaired recombinase activity in OS severely affects expression of the pre-T-cell receptor complex in immature thymocytes, which is crucial for an efficient development of the thymic epithelial component. Anti-CD3ε monoclonal antibody (mAb) treatment in RAG2(-/-) mice was previously shown to mimic pre-TCR signaling promoting thymic expansion. Here we show the effect of anti-CD3ε mAb administration in the RAG2(R229Q) mouse model, which closely recapitulates human OS. These animals, in spite of the inability to induce the autoimmune regulator, displayed a significant amelioration in thymic epithelial compartment and an important reduction of peripheral T-cell activation and tissue infiltration. Furthermore, by injecting a high number of RAG2(R229Q) progenitors into RAG2(-/-) animals previously conditioned with anti-CD3ε mAb, we detected autoimmune regulator expression together with the absence of peripheral immunopathology. These observations indicate that improving epithelial thymic function might ameliorate the detrimental behavior of the cell-autonomous RAG defect. Our data provide important therapeutic proof of concept for future clinical applications of anti-CD3ε mAb treatment in severe combined immunodeficiency forms characterized by poor thymus function and autoimmunity.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Autoimmune Diseases/prevention & control , CD3 Complex/immunology , Severe Combined Immunodeficiency/therapy , Thymus Gland/drug effects , Animals , Animals, Newborn , Autoimmunity/drug effects , Autoimmunity/genetics , DNA-Binding Proteins/genetics , Disease Models, Animal , Gene Knock-In Techniques , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Size/drug effects , Severe Combined Immunodeficiency/immunology , Severe Combined Immunodeficiency/pathology , Thymus Gland/immunology , Thymus Gland/pathology , Thymus Gland/ultrastructureABSTRACT
There are thousands of rare genetic diseases that could be treated with classical gene therapy strategies such as the addition of the defective gene via viral or non-viral delivery or by direct gene editing. However, several genetic defects are too complex for these approaches. These "genomic mutations" include aneuploidies, intra and inter chromosomal rearrangements, large deletions, or inversion and copy number variations. Chromosome transplantation (CT) refers to the precise substitution of an endogenous chromosome with an exogenous one. By the addition of an exogenous chromosome and the concomitant elimination of the endogenous one, every genetic defect, irrespective of its nature, could be resolved. In the current review, we analyze the state of the art of this technique and discuss its possible application to human pathology. CT might not be limited to the treatment of human diseases. By working on sex chromosomes, we showed that female cells can be obtained from male cells, since chromosome-transplanted cells can lose either sex chromosome, giving rise to 46,XY or 46,XX diploid cells, a modification that could be exploited to obtain female gametes from male cells. Moreover, CT could be used in veterinary biology, since entire chromosomes containing an advantageous locus could be transferred to animals of zootechnical interest without altering their specific genetic background and the need for long and complex interbreeding. CT could also be useful to rescue extinct species if only male cells were available. Finally, the generation of "synthetic" cells could be achieved by repeated CT into a recipient cell. CT is an additional tool for genetic modification of mammalian cells.
Subject(s)
Chromosomes , Genomic Medicine , Animals , Humans , Genetic Therapy/methods , Male , Female , Synthetic Biology/methodsABSTRACT
The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.
Subject(s)
Osteoblasts/cytology , Osteoblasts/metabolism , Osteogenesis Imperfecta/metabolism , Adipogenesis/drug effects , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Autophagy/drug effects , Blotting, Western , Boronic Acids/pharmacology , Bortezomib , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Flow Cytometry , Immunohistochemistry , Mice , Osteogenesis/drug effects , Osteogenesis Imperfecta/pathology , Pyrazines/pharmacologyABSTRACT
Since its identification, the RANKL cytokine has been demonstrated to play a crucial role in bone homeostasis and lymphoid tissue organization. Genetic defects impairing its function lead to a peculiar form of autosomal recessive osteopetrosis (ARO), a rare genetic bone disease presenting early in life and characterized by increased bone density due to failure in bone resorption by the osteoclasts. Hematopoietic stem cell transplantation (HSCT) is the only option for the majority of patients affected by this life-threatening disease. However, the RANKL-dependent ARO does not gain any benefit from this approach, because the genetic defect is not intrinsic to the hematopoietic osteoclast lineage but rather to the mesenchymal one. Of note, we recently provided proof of concept of the efficacy of a pharmacological RANKL-based therapy to cure this form of the disease. Here we provide an overview of the diverse roles of RANKL in the bone and immune systems and review the clinical features of RANKL-deficient ARO patients and the results of our preclinical studies. We emphasize that these patients present a continuous worsening of the disease in the absence of a cure and strongly wish that the therapy we propose will be further developed.
Subject(s)
Bone Resorption/drug therapy , Bone and Bones/drug effects , Osteoclasts/drug effects , Osteopetrosis/drug therapy , RANK Ligand/immunology , RANK Ligand/pharmacology , Animals , Bone Density/drug effects , Bone Resorption/genetics , Bone Resorption/immunology , Bone Resorption/pathology , Bone and Bones/immunology , Bone and Bones/pathology , Gene Expression Regulation/immunology , Genes, Recessive , Hematopoietic Stem Cell Transplantation , Homeostasis/drug effects , Homeostasis/genetics , Humans , Immune System/drug effects , Mice , Mutation , Osteoclasts/immunology , Osteoclasts/pathology , Osteopetrosis/genetics , Osteopetrosis/immunology , Osteopetrosis/pathology , RANK Ligand/geneticsABSTRACT
BACKGROUND AND OBJECTIVE: The autofluorescence (AF) analysis allows in vivo, real-time assessment of cell functional activities, depending on the presence of biomolecules strictly involved in metabolic reactions and acting as endogenous fluorophores. Pluripotent stem cells during differentiation are known to undergo changes in their morphofunctional properties, with particular reference to bioenergetic metabolic signatures involving endogenous fluorophores such as NAD(P)H, flavins, lipofuscin-like lipopigments. Since the development of regenerative therapies based on pluripotent cells requires a careful monitoring of the successful maturation into the desired phenotype, aim of our work is to evaluate the AF potential to assess the differentiation phases in a murine stem cell model. STUDY DESIGN/MATERIALS AND METHODS: Mouse embryonic stem cells (ESCs) maintained with and without leukemia inhibitory factor (LIF), embryoid bodies (EBs), and EB-derived cells undergoing spontaneous differentiation toward the hematopoietic lineage have been used as a sample models. Cell AF properties have been characterized upon 366-nm excitation, under living conditions and in the absence of exogenous markers. Imaging, microspectrofluorometric techniques, and spectral fitting analysis based on the spectral parameters of each endogenous fluorophore have been applied to estimate their contribution to the whole cell AF emission spectra. Specific cytochemical labeling has been performed to validate AF data. RESULTS: Depending on the differentiation phases, cells undergo changes in morphology, AF distribution patterns, and AF emission spectral profiles. These latter reflect variations in the single endogenous fluorophore contribution to the overall emission. The coenzyme NAD(P)H accounts for up to 80% of the whole spectral area. The free form prevails on the bound one, and their changes have been investigated in terms of NAD(P)Hbound/free and redox ratios. These values vary in agreement with a slow metabolic activity and prevailing glycolytic metabolism in the undifferentiated HM1 cells, an increased metabolic activity still relying on glycolysis during the early differentiation phases, and an increased oxidative phosphorylation in EB and hematopoietic precursor cells. Lipofuscin-like lipopigments decrease following differentiation, and porphyrins contributing for less than 5%, prevail in the more actively differentiating cells. These results reflect the shift between anaerobic and aerobic respiration following differentiation, consistently with a decreased autophagy of cell organelles (i.e., mitochondria, as a strategy reported in the literature to keep the undifferentiated homeostasis state), higher mitochondrial activity with more numerous NADH binding sites and synthesis of heme as prosthetic group of proteins, that is, cytochromes. CONCLUSIONS: These data open promising perspectives for the monitoring of stem cells differentiation under living conditions without labeling with exogenous agents (inducing perturbations when used in vivo), or immunomarkers not always available for veterinary and zootechnics, by exploiting endogenous fluorophores as intrinsic biomarkers of cell morphofunctional changes.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Optical Imaging , Animals , Biomarkers/chemistry , Biomarkers/metabolism , Cell Respiration , Embryonic Stem Cells/metabolism , Mice , Microspectrophotometry , NADP/chemistry , NADP/metabolism , Spectrometry, FluorescenceABSTRACT
Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Mutations in the CLCN7 gene are responsible not only for a substantial portion of ARO patients but also for other forms of osteopetrosis characterized by different severity and inheritance. The lack of a clear genotype/phenotype correlation makes genetic counseling a tricky issue for CLCN7-dependent osteopetrosis. Here, we characterize the first homozygous interstitial deletion in 16p13.3, detected by array comparative genomic hybridization in an ARO patient of Jordanian origin. The deletion involved other genes besides CLCN7, while the proband displayed a classic ARO phenotype; however, her early death did not allow more extensive clinical investigations. The identification of this novel genomic deletion involving a large part of the CLCN7 gene is of clinical relevance, especially in prenatal diagnosis, and suggests the possibility that this kind of mutation has been underestimated so far. These data highlight the need for alternative approaches to genetic analysis also in other ARO-causative genes.
Subject(s)
Chromosomes, Human, Pair 16/genetics , Gene Deletion , Genes, Recessive , Homozygote , Osteopetrosis/genetics , Base Sequence , Chloride Channels/genetics , Comparative Genomic Hybridization , Humans , Infant , Molecular Sequence Data , Mutation , PhenotypeABSTRACT
Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous developmental disorder characterized by facial dysmorphia, upper limb malformations, growth and cognitive retardation. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A and SMC3 are present in approximately 65% of CdLS patients. In addition to their canonical roles in chromosome segregation, the cohesin proteins are involved in other biological processes such as regulation of gene expression, DNA repair and maintenance of genome stability. To gain insights into the molecular basis of CdLS, we analyzed the affinity of mutated SMC1A and SMC3 hinge domains for DNA. Mutated hinge dimers bind DNA with higher affinity than wild-type proteins. SMC1A- and SMC3-mutated CdLS cell lines display genomic instability and sensitivity to ionizing radiation and interstrand crosslinking agents. We propose that SMC1A and SMC3 CdLS mutations affect the dynamic association between SMC proteins and DNA, providing new clues to the underlying molecular cause of CdLS.
Subject(s)
Cell Cycle Proteins/metabolism , Chondroitin Sulfate Proteoglycans/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , De Lange Syndrome/genetics , Mutation , Cell Cycle Proteins/genetics , Cell Line , Cells, Cultured , Chondroitin Sulfate Proteoglycans/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA/genetics , DNA/metabolism , DNA Damage , DNA-Binding Proteins/genetics , De Lange Syndrome/metabolism , Female , Humans , Protein Binding , CohesinsABSTRACT
Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.
Subject(s)
Agammaglobulinemia/blood , Osteoclasts/pathology , Osteopetrosis/genetics , Receptor Activator of Nuclear Factor-kappa B/genetics , Acid Phosphatase/metabolism , Actins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Argentina , Arginine/metabolism , Biopsy , Case-Control Studies , Cell Line, Transformed , Cell Proliferation , Cell Transformation, Viral , Cells, Cultured , Cohort Studies , Consanguinity , Cysteine/metabolism , DNA Mutational Analysis , Dendrites/physiology , Female , Genes, Recessive , Herpesvirus 4, Human/physiology , Heterozygote , Homozygote , Humans , Ilium/surgery , Isoenzymes/metabolism , Leukocyte Common Antigens/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/pathology , Lipopolysaccharides/pharmacology , Macrophage Colony-Stimulating Factor/pharmacology , Male , Models, Immunological , Molecular Sequence Data , Mutation, Missense , Osteoclasts/metabolism , Osteoclasts/ultrastructure , Osteopetrosis/diagnosis , Osteopetrosis/diagnostic imaging , Osteopetrosis/pathology , Osteopetrosis/physiopathology , Osteoprotegerin/metabolism , Pakistan , Pedigree , Polymorphism, Genetic , Protein Structure, Tertiary , RANK Ligand/metabolism , Radiography, Thoracic/methods , Receptor Activator of Nuclear Factor-kappa B/chemistry , Receptor Activator of Nuclear Factor-kappa B/immunology , Receptors, Vitronectin/metabolismABSTRACT
Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.
Subject(s)
Aging/physiology , Bone Marrow Transplantation/methods , Fetal Research , Osteogenesis Imperfecta/prevention & control , Osteogenesis Imperfecta/therapy , Uterus/physiology , Animals , Bone Marrow Cells/cytology , Collagen/metabolism , Disease Models, Animal , Extracellular Space/chemistry , Female , Gene Knock-In Techniques , Genes, Dominant , Graft Survival , Mice , Mice, Transgenic , Osteogenesis Imperfecta/metabolism , Osteogenesis Imperfecta/pathology , Phenotype , Pregnancy , Spectrum Analysis, Raman , Survival Rate , Tissue DonorsABSTRACT
The underlying mechanism by which anti-VEGF agents prolong cancer patient survival is poorly understood. We show that in a mouse tumor model, VEGF systemically impairs functions of multiple organs including those in the hematopoietic and endocrine systems, leading to early death. Anti-VEGF antibody, bevacizumab, and anti-VEGF receptor 2 (VEGFR-2), but not anti-VEGFR-1, reversed VEGF-induced cancer-associated systemic syndrome (CASS) and prevented death in tumor-bearing mice. Surprisingly, VEGFR2 blockage improved survival by rescuing mice from CASS without significantly compromising tumor growth, suggesting that "off-tumor" VEGF targets are more sensitive than the tumor vasculature to anti-VEGF drugs. Similarly, VEGF-induced CASS occurred in a spontaneous breast cancer mouse model overexpressing neu. Clinically, VEGF expression and CASS severity positively correlated in various human cancers. These findings define novel therapeutic targets of anti-VEGF agents and provide mechanistic insights into the action of this new class of clinically available anti-VEGF cancer drugs.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents/therapeutic use , Neoplasms, Experimental/drug therapy , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Anemia/physiopathology , Animals , Capillary Permeability , Humans , Immunohistochemistry , Liver/physiopathology , Mice , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/physiopathology , Neoplasms, Experimental/prevention & controlABSTRACT
BACKGROUND: Omenn syndrome (OS) is an autosomal-recessive disorder characterized by severe immunodeficiency and T-cell-mediated autoimmunity. The disease is caused by hypomorphic mutations in recombination-activating genes that hamper the process of Variable (V) Diversity (D) Joining (J) recombination, leading to the generation of autoreactive T cells. We have previously shown that in OS the expression of autoimmune regulator, a key factor governing central tolerance, is markedly reduced. OBJECTIVE: Here, we have addressed the role of peripheral tolerance in the disease pathogenesis. METHODS: We have analyzed forkhead box protein P3 (FOXP3) expression in peripheral blood T cells of 4 patients with OS and in lymphoid organs of 8 patients with OS and have tested the suppressive activity of sorted CD4(+) CD25(high) peripheral blood T cells in 2 of these patients. RESULTS: We have observed that CD4(+)CD25(high)T cells isolated ex vivo from patients with OS failed to suppress proliferation of autologous or allogenic CD4(+) responder T cells. Moreover, despite individual variability in the fraction of circulating FOXP3(+) CD4 cells in patients with OS, the immunohistochemical analysis of FOXP3 expression in lymph nodes and thymus of patients with OS demonstrated a severe reduction of this cell subset compared with control tissues. CONCLUSION: Overall, these results suggest a defect of regulatory T cells in OS leading to a breakdown of peripheral tolerance, which may actively concur to the development of autoimmune manifestations in the disease.
Subject(s)
Forkhead Transcription Factors/metabolism , Severe Combined Immunodeficiency/immunology , Severe Combined Immunodeficiency/physiopathology , T-Lymphocytes, Regulatory/pathology , Autoimmune Diseases/immunology , Autoimmune Diseases/physiopathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Down-Regulation , Forkhead Transcription Factors/genetics , Homeodomain Proteins/genetics , Humans , Infant, Newborn , Interleukin-2 Receptor alpha Subunit/metabolism , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Severe Combined Immunodeficiency/genetics , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolismABSTRACT
PBX1 regulates the balance between self-renewal and differentiation of hematopoietic stem cells and maintains proto-oncogenic transcriptional pathways in early progenitors. Its increased expression was found in myeloproliferative neoplasm (MPN) patients bearing the JAK2V617F mutation. To investigate if PBX1 contributes to MPN, and to explore its potential as therapeutic target, we generated the JP mouse strain, in which the human JAK2 mutation is induced in the absence of PBX1. Typical MPN features, such as thrombocythemia and granulocytosis, did not develop without PBX1, while erythrocytosis, initially displayed by JP mice, gradually resolved over time; splenic myeloid metaplasia and in vitro cytokine independent growth were absent upon PBX1 inactivation. The aberrant transcriptome in stem/progenitor cells from the MPN model was reverted by the absence of PBX1, demonstrating that PBX1 controls part of the molecular pathways deregulated by the JAK2V617F mutation. Modulation of the PBX1-driven transcriptional program might represent a novel therapeutic approach.
Subject(s)
Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cells/metabolism , Myeloproliferative Disorders/genetics , Neoplasms/genetics , Pre-B-Cell Leukemia Transcription Factor 1/genetics , Animals , Disease Models, Animal , Disease Progression , Gene Expression Profiling/methods , Humans , Mice, Knockout , Mice, Transgenic , Mutation , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , Neoplasms/metabolism , Neoplasms/pathology , Pre-B-Cell Leukemia Transcription Factor 1/metabolism , RNA-Seq/methods , Signal Transduction/geneticsABSTRACT
The "Osteopetroses" are genetic diseases whose clinical picture is caused by a defect in bone resorption by osteoclasts. Three main forms can be distinguished on the basis of severity, age of onset and means of inheritance: the dominant benign, the intermediate and the recessive severe form. While several genes have been involved in the pathogenesis of the different types of osteopetroses, the CLCN7 gene has drawn the attention of many researchers, as mutations within this gene are associated with very different phenotypes. We report here the characterization of 25 unpublished patients which has resulted in the identification of 20 novel mutations, including 11 missense mutations, 6 causing premature termination, 1 small deletion and 2 putative splice site defects. Careful analysis of clinical and molecular data led us to several conclusions. First, intermediate osteopetrosis is not homogeneous, since it can comprise both severe dominant forms with an early onset and recessive ones without central nervous system involvement. Second, the appropriateness of haematopoietic stem cell transplantation in CLCN7-dependent ARO patients has to be carefully evaluated and exhaustive CNS examination is strongly suggested, as transplantation can almost completely cure the disease in situations where no primary neurological symptoms are present. Finally, the analysis of this largest cohort of CLCN7-dependent ARO patients together with some ADO II families allowed us to draw preliminary genotype-phenotype correlations suggesting that haploinsufficiency is not the mechanism causing ADO II. The availability of biochemical assays to characterize ClC-7 function will help to confirm this hypothesis.
Subject(s)
Chloride Channels/genetics , Mutation , Osteopetrosis/genetics , Osteopetrosis/physiopathology , Severity of Illness Index , Adult , Age of Onset , Child , Child, Preschool , Chloride Channels/chemistry , Chloride Channels/metabolism , Crystallization , Female , Genotype , Humans , Infant , Infant, Newborn , Male , Models, Molecular , PhenotypeABSTRACT
Rag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2(R229Q/R229Q) mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2(R229Q/R229Q) mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.